All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sciatic Nerve Extraction
<ol>
	<li>Take mice whose sciatic nerves are previously injected with cancer cells and euthanize them with CO2. Place the mouse on its ventral side and stabilize the distal limbs using pins.</li>
	<li>Remove the skin on the dorsal side of the injected limb and torso.</li>
	<li>Using blunt dissection, expose the sciatic nerve deep into the muscles.</li>
	<li>The nerve courses between the ilium and the sacrum. To have access to the nerve at the spinal cord region, separate the two bones. First, insert closed scissors in the narrow area where the sciatic nerve is located, and then open the scissors while holding the mouse. Be delicate and maintain the integrity of the sciatic nerve during the dissection.</li>
	<li>Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord. 
	NOTE: Invaded nerves are extremely fragile and prone to breaking under tension or forceful handling.</li>
	<li>Harvest the nerve by first cutting its distal end. Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end, as close as possible to its exit from the spinal column.</li>
	<li>Record then gross length of invasion using a Vernier caliper. This macroscopic estimation is only indicative.</li>
</ol>
2. Nerve Processing and Quantification
<ol>
	<li>Embed the dissected nerve in OCT compound. Make sure to place nerves longitudinally and as flat on the bottom of the mold as possible.</li>
	<li>Indicate on the cassette the proximal and distal side of the nerve by marking the letter P (Proximal) and D (Distal).</li>
	<li>Place embedded nerves on top of dry ice, if they are not sectioned immediately. Sample could be preserved at -80 &#176;C for several weeks.</li>
	<li>Section samples using a Cryostat microtome at 5 &#956;m thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve.</li>
	<li>Stain slides using H&amp;E staining.</li>
	<li>Digitally scan stained nerve sections with a slide-scanner that provides high-resolution digital data.</li>
	<li>Using imaging software, quantify the length of invasion by clicking the measure distance button, area of invasion, or other desired parameters. For a good estimation of the length of invasion, use multiple sections (2 to 4) of the same nerve.</li>
</ol>

<table><tbody><tr><td>Mouse</td><td></td><td></td><td>Number and age variable depending on experimental needs</td></tr><tr><td>Sterile surgical tools (scissors and forceps)</td><td></td><td></td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>&amp;nbsp;VWR</td><td>&amp;nbsp;25608-930&amp;nbsp;</td><td></td></tr><tr><td>Tissue-Tek Cryomold Molds</td><td>&amp;nbsp;VWR</td><td>&amp;nbsp;25608-916</td><td></td></tr></tbody></table>

murine sciatic nerve model of perineural invasion or pni: extraction and processing of sciatic nerve to understand invasion by cancer cells

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells

Source: Deborde, S. et al. <a href='https://www.jove.com/video/56857' target='_blank'>An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure to establish a murine model of perineural invasion by injecting cancer cells into the sciatic nerve. The model can help gain an understanding of the magnitude of nerve invasion for subsequent assessment of anti-cancer drugs.

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we describe the procedure to ...

Cancer cells can move from their site of origin and invade the surrounding nerves in a process called perineural invasion or PNI. PNI is observed in an animal model by injecting cancer cells into a major nerve such as the sciatic nerve, which is then extracted to examine the extent of invasion.
To begin, prep a euthanized mouse previously injected with cancer cells in the prone position. Remove the sutures from the hindlimb - the site of tumor cell injection to expose the sciatic nerve that innervates the hindlimb muscles.
Use scissors to separate the muscles and expose the underlying sciatic nerve. Dissect the nerve ends to isolate it from the body. Now, use a caliper to measure and record the macroscopic extent of cancer cell invasion.
Next, place the dissected nerve in an embedding medium to enable sample preparation at low temperatures. Using a cryostat microtome, cut thin sections of the embedded nerve and place them on a slide. Stain the slide with a hematoxylin-eosin histological stain.
Hematoxylin - a basic dye - stains acidic components like the nuclei, while eosin - an acidic dye - stains basic components like the cytoplasm. Observe the slide under a microscope to measure the length and area of neural invasion.

On postoperative day 7, place the euthanized mouse on its ventral side and stabilize the distal limbs using pins. After removing the skin on the dorsal side of the injected limb and torso, use blunt dissection to expose the sciatic nerve deep to the muscles.
To access the nerve at the spinal cord region, separate the ilium and the sacrum by inserting closed scissors in the narrow area where the sciatic nerve is located, then open the scissors while holding the mouse. Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord.
Careful handling is essential as invaded nerves are extremely fragile and prone to breaking under tension or forceful handling. Be delicate and maintain the integrity of the sciatic nerve during the dissection.
Harvest the nerve by first cutting its distal end. Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end as close as possible to its exit from the spinal column. Use a vernier caliper to estimate the gross length of invasion. Begin processing by embedding the dissected nerve in OCT compound. Place nerves longitudinally and as flat on the bottom of the mold as possible.
Indicate on the cassette the proximal and distal side of the nerve by marking the letter P and D. Then, place the embedded nerves on top of dry ice. Section samples using a Cryostat microtome at 5-micron thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve. After staining the slides with H&amp;E and obtaining images with a slide scanner, use imaging software to quantify the length of invasion.

murine-sciatic-nerve-model-perineural-invasion-or-pni-extraction

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

Ex vivo studies

3.6K Views. Cancer cells can move from their site of origin and invade the surrounding nerves in a process called perineural invasion or PNI. PNI is observed in an animal model by injecting cancer cells into a major nerve such as the sciatic nerve, which is then extracted to examine the extent of invasion. To begin, prep a euthanized mouse previously injected with cancer cells in the prone position. Remove the sutures from the hindlimb - the site of tumor cell injection to expose the sciatic nerve...

Abstract

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sciatic Nerve Extraction
<ol>
	<li>Take mice whose sciatic nerves are previously injected with cancer cells and euthanize them with CO2. Place the mouse on its ventral side and stabilize the distal limbs using pins.</li>
	<li>Remove the skin on the dorsal side of the injected limb and torso.</li>
	<li>Using blunt dissection, expose the sciatic nerve deep into the muscles.</li>
	<li>The nerve courses between the ilium and the sacrum. To have access to the nerve at the spinal cord region, separate the two bones. First, insert closed scissors in the narrow area where the sciatic nerve is located, and then open the scissors while holding the mouse. Be delicate and maintain the integrity of the sciatic nerve during the dissection.</li>
	<li>Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord. 
	NOTE: Invaded nerves are extremely fragile and prone to breaking under tension or forceful handling.</li>
	<li>Harvest the nerve by first cutting its distal end. Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end, as close as possible to its exit from the spinal column.</li>
	<li>Record then gross length of invasion using a Vernier caliper. This macroscopic estimation is only indicative.</li>
</ol>
2. Nerve Processing and Quantification
<ol>
	<li>Embed the dissected nerve in OCT compound. Make sure to place nerves longitudinally and as flat on the bottom of the mold as possible.</li>
	<li>Indicate on the cassette the proximal and distal side of the nerve by marking the letter P (Proximal) and D (Distal).</li>
	<li>Place embedded nerves on top of dry ice, if they are not sectioned immediately. Sample could be preserved at -80 &#176;C for several weeks.</li>
	<li>Section samples using a Cryostat microtome at 5 &#956;m thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve.</li>
	<li>Stain slides using H&E staining.</li>
	<li>Digitally scan stained nerve sections with a slide-scanner that provides high-resolution digital data.</li>
	<li>Using imaging software, quantify the length of invasion by clicking the measure distance button, area of invasion, or other desired parameters. For a good estimation of the length of invasion, use multiple sections (2 to 4) of the same nerve.</li>
</ol>

Protocol

Disclosures

<table><tbody><tr><td>Mouse</td><td></td><td></td><td>Number and age variable depending on experimental needs</td></tr><tr><td>Sterile surgical tools (scissors and forceps)</td><td></td><td></td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>&nbsp;VWR</td><td>&nbsp;25608-930&nbsp;</td><td></td></tr><tr><td>Tissue-Tek Cryomold Molds</td><td>&nbsp;VWR</td><td>&nbsp;25608-916</td><td></td></tr></tbody></table>

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number	Comments
Mouse			Number and age variable depending on experimental needs
Sterile surgical tools (scissors and forceps)
Tissue-Tek O.C.T. Compound	VWR	25608-930
Tissue-Tek Cryomold Molds	VWR	25608-916